Bacterial resistance to antibiotics has seriously limited our capacity to overcome infectious disease. Cases of resistance have emerged in virtually all hospital-acquired pathogen-antimicrobial combinations. Soon our most serious infectious threats will be untreatable given our dwindling arsenal of effective antibiotics. Ramoplanins and Enduracidins comprise a family of peptide antibiotics with potent broad-spectrum activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium (VRE), and cephalosporin-resistant Streptococcus pneumonia, three important opportunistic human pathogens. Ramoplanin and Enduracidin inhibit the transglycosylation cross-linking step of bacterial peptidoglycan biosynthesis in a complex mechanism involving capture of the transglycosylase substrate, Lipid Intermediate II. In Aim 1 we seek to determine the molecular mechanisms of a recently identified secondary mechanism of Ramoplanin, inhibition of cell wall protein anchoring and biofilm development in Gram-positive bacteria. Inhibition of cell wall protein anchoring diminishes virulence potential and results in an impaired capacity to form persistent infections. In Aim 2 we will proactively characterize the molecular mechanisms of Ramoplanin resistance in Gram-positive bacteria. Lastly, in Aim 3 we will perform structural analyses of Enduracidin and the Enduracidin::Lipid II complex in solution and in membrane environments using solution and solid state NMR approaches. In turn, we hope to apply the knowledge gained to the development of alternative antibiotics with improved activity against infections due to resistant and virulent bacterial phenotypes.